Antimicrobial peptides (APs), including defensins and cathelicidins, are an important component of the innate immune system for combating bacteria and other microbial invaders. Many Gram-negative pathogens utilize the Sensitive to Antimicrobial Peptides (Sap) transporter to survive attack by APs during infection. The Sap transporter has been shown to confer resistance to a wide variety of APs and contributes significantly to virulence. Thus, the Sap transporter is an excellent candidate for targeting development of novel antimicrobial therapeutics that would likely be effective against many Gram-negative pathogens. However, little is known about how the transporter functions with APs to confer resistance to their killing effects. The long-term goal of this project is to understand the mechanism of action of the Sap transporter so that novel antimicrobial agents can be developed that target this transporter. Using the Sap transporter of Haemophilus ducreyi as a model system, we have begun to study the role of specific Sap proteins in conferring AP resistance. H. ducreyi is the causative agent of chancroid, a sexually transmitted genital ulcer disease that facilitates transmission and acquisition of human immunodeficiency virus (HIV) and contributes to the spread of HIV in areas with endemic chancroid. H. ducreyi is one of few pathogens amenable to human inoculation experiments, so studies of the Sap transporter and its interactions with APs can be directly extended to human disease. Our preliminary data with the Sap transporter of H. ducreyi raise questions about the proposed model of the mechanism of Sap transporter activity, including the role of the periplasmic peptide binding component, SapA, in transporter activity and in defining the specificity of the transporter for certain APs. In this proposal, we will establish methods to directly examine the effects of the Sap transporter on the human cathelicidin AP LL37 and use these methods to determine the role of SapA in transporter activity, define possible additional proteins involved in transporter function, and determine which components of the transporter confer specificity for transport of only certain APs. Outcomes of these studies provide the tools required to study the mechanism of action of the Sap transporter and will define the repertoire of proteins that interact specifically with APs for Sap-mediated transport. Data from this proposal will thus provide the methodologies and preliminary data for an R01 application to study in greater detail the interactions of the Sap transporter with APs, which is necessary for developing antimicrobials that target this important virulence factor.